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PubMed1991

Analysis of cDNA clones for Acanthamoeba profilin-I and profilin-II shows end to end homology with vertebrate profilins and a small family of profilin genes.

We have cloned and sequenced full length cDNAs for Acanthamoeba profilin-I and profilin-II. The genes and the encoded proteins are nearly identical except for the region between bp 121 and 210 where 35% of the nucleotides and 47% of amino acids differ. Most of these substitutions are conservative, although three of them are responsible for the differences in the isoelectric points of the isoforms [Kaiser et al., Cell Biol., 102:221-226, 1986]. The DNA sequence revealed six corrections in the previously published protein sequence of profilin-I [Ampe et al., J. Biol. Chem. 260:834-840, 1985] and for the first time resolved the ambiguities at the five positions where profilin-IA and -IB differ. The DNA sequence of profilin-II also allowed us to make two corrections in the protein sequence [Ampe et al., FEBS Lett. 228:17-21, 1988a]. Probes prepared from the cDNAs revealed 1 profilin-IA gene, one strongly cross-hybridizing profilin-I gene and one strongly reacting profilin-II gene on Southern blots of Acanthamoeba DNA. Weaker reactions with other genomic DNA fragments leave open the possibility of one additional gene each for profilin-I and profilin-II. Four different profilin RNAs were resolved on Northern blots. It possible to align the sequences of the three Acanthamoeba profilins with the sequences of nine other profilins from five different phyla. There are only two invariant residues in these profilin sequences, but many pairwise identities and conservative substitutions that indicate considerable divergence of this family of proteins from its ancestral precursor.

Acanthamoeba↗

PubMed1996

Arabidopsis profilins are functionally similar to yeast profilins: identification of a vascular bundle-specific profilin and a pollen-specific profilin.

Four members of the Arabidopsis profilin (pfn) multigene family have been cloned, sequenced and analyzed. By RNA gel blot analysis it has been shown that these four genes fall into two groups: one group (pfn1 and pfn2) is expressed in all organs of the plant and the other group (pfn3 and pfn4) in floral tissues only. Based on amino acid sequence alignment Arabidopsis profilins can be divided into the same two groups: PFN1 and PFN2 are 89% identical and PFN3 and PFN4 are 91% identical. Between these two groups they are 71-75% identical. The Arabidopsis profilins bind poly-L-proline and can complement both the Saccharomyces cerevisiae profilin deletion mutant and the Schizosaccharomyces pombe cdc3-124/profilin mutation, showing that the plant profilins are functionally similar to yeast profilins despite the low amino acid sequence homology. Analysis of pfn promoter-GUS fusion genes in transgenic Arabidopsis shows that pfn2 is specifically expressed in the vascular bundles of roots, hypocotyls, cotyledons, leaves, sepals, petals, stamen filaments and stalks of developing seeds, whereas expression of pfn4 is restricted to mature and germinating pollen grains.

Amino Acid Sequence↗

PubMed1997

Effects of profilin-annexin I association on some properties of both profilin and annexin I: modification of the inhibitory activity of profilin on actin polymerization and inhibition of the self-association of annexin I and its interactions with liposomes.

We have previously shown that annexin I, a member of a family of calcium-dependent phospholipid and membrane binding proteins, interacts with profilin with high specificity and affinity. This finding further suggests that annexin I is involved through profilin in the regulation of membrane-cytoskeleton organization. We have investigated the consequences of a complex formed by these two proteins on the functions of both profilin and annexin I. Annexin I is able to modify the inhibitory effect of profilin on actin polymerization. This action is partial and the mechanism involved appears to be complex. On the other hand, the association between annexin I and profilin is sufficiently strong to inhibit the self-association of annexin I. The binding capacity of annexin I to liposomes containing phosphatidylserine, which mimics annexin I binding to membranes, is also decreased by profilin. This binding is nevertheless restored when phosphatidylinositol 4,5-biphosphate (PtdInsP2) is included in the liposomes. Finally, the capacity of annexin I to aggregate liposomes is also modified. It is worthwhile mentioning that the liposomes-binding and liposomes-aggregating activities of annexin I are independently regulated. The cell localization and functions of annexin I and profilin suggest that interaction between these two proteins may be directly implicated in the regulation of membrane-cytoskeleton. The phospholipid composition of membranes may be one of the modulating factors.

Actins↗

PubMed1994

cDNA cloning and expression of timothy grass (Phleum pratense) pollen profilin in Escherichia coli: comparison with birch pollen profilin.

Profilin, an actin-binding protein, was previously described as a ubiquitous allergen which is responsible for cross-reactivities in about 20% of pollen and food allergic patients. A complete cDNA clone coding for timothy grass (Phelum pratense) pollen profilin was isolated using allergic patients IgE. The deduced amino acid sequence of timothy grass profilin shares a sequence identity of 79% with birch profilin and other plant profilins and a lower average sequence identity of 35% with other eukaryotic profilins. The high degree of homology among different plant profilins at the DNA and protein level explains the extensive cross-reactivities observed in profilin allergic patients. Recombinant timothy grass pollen profilin was expressed in Escherichia coli as a beta-galactosidase fusion protein and shown to bind IgE from profilin allergic patients similar to recombinant birch profilin. Slight differences regarding the IgE-binding capacity of birch and timothy grass profilin indicate that not all IgE-epitopes of the two profilins are conserved. It is speculated that profilin allergic patients were initially sensitized against a certain profilin and then cross-react with the homologous proteins.

Allergens↗

PubMed1996

Plant profilins rescue the aberrant phenotype of profilin-deficient Dictyostelium cells.

To characterize the function of plant profilins in vivo, we expressed two pollen specific Zea mays (maize) profilin isoforms in profilin-minus Dictyostelium discoideum mutants. In maize, profilins exist as a multigene family containing 4 or more members which are highly similar to each other but substantially less similar to profilins from animals and lower eukaryotes. Previously we have shown that D. discoideum profilin-minus cells have an aberrant phenotype due to defects in cell shape, cytokinesis, and development. These defects could be rescued by introducing the pollen-specific profilins 1 or 2 from maize using a newly constructed expression vector. Expression of the heterologous profilins in Dictyostelium clones was assayed by affinity purification of the pollen profilins with poly-proline agarose and by immunoblotting with a polyclonal antiserum raised against maize pollen profilin. In contrast to the profilin-minus mutants, Dictyostelium cells expressing plant profilins showed normal cell shape, contained less F-actin, and were able to form fruiting bodies. These data provide genetic evidence that maize pollen profilins, even though they are specific for a distinct developmental stage, share functional properties with profilin from a lower eukaryote and apparently act as G-actin-sequestering proteins in this system.

Animals↗

PubMed1989

Acanthamoeba actin and profilin can be cross-linked between glutamic acid 364 of actin and lysine 115 of profilin.

Acanthamoeba profilin was cross-linked to actin via a zero-length isopeptide bond using carbodiimide. The covalently linked 1:1 complex was purified and treated with cyanogen bromide. This cleaves actin into small cyanogen bromide (CNBr) peptides and leaves the profilin intact owing to its lack of methionine. Profilin with one covalently attached actin CNBr peptide was purified by gel filtration followed by gel electrophoresis and electroblotting on polybase-coated glass-fiber membranes. Since the NH2 terminus of profilin is blocked, Edman degradation gave only the sequence of the conjugated actin CNBr fragment beginning with Trp-356. The profilin-actin CNBr peptide conjugate was digested further with trypsin and the cross-linked peptide identified by comparison with the tryptic peptide pattern obtained from carbodiimide-treated profilin. Amino-acid sequence analysis of the cross-linked tryptic peptides produced two residues at each cycle. Their order corresponds to actin starting at Trp-356 and profilin starting at Ala-94. From the absence of the phenylthiohydantoin-amino acid residues in specific cycles, we conclude that actin Glu-364 is linked to Lys-115 in profilin. Experiments with the isoforms of profilin I and profilin II gave identical results. The cross-linked region in profilin is homologous with sequences in the larger actin filament capping proteins fragmin and gelsolin.

Acanthamoeba↗

PubMed1998

In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly.

Profilins are thought to be essential for regulation of actin assembly. However, the functions of profilins in mammalian tissues are not well understood. In mice profilin I is expressed ubiquitously while profilin II is expressed at high levels only in brain. In extracts from mouse brain, profilin I and profilin II can form complexes with regulators of endocytosis, synaptic vesicle recycling and actin assembly. Using mass spectrometry and database searching we characterized a number of ligands for profilin I and profilin II from mouse brain extracts including dynamin I, clathrin, synapsin, Rho-associated coiled-coil kinase, the Rac-associated protein NAP1 and a member of the NSF/sec18 family. In vivo, profilins co-localize with dynamin I and synapsin in axonal and dendritic processes. Our findings strongly suggest that in brain profilin I and profilin II complexes link the actin cytoskeleton and endocytic membrane flow, directing actin and clathrin assembly to distinct membrane domains.

Actins↗

PubMed1996

The Arabidopsis profilin gene family. Evidence for an ancient split between constitutive and pollen-specific profilin genes.

Profilin is a ubiquitous eukaryotic protein that regulates the actin cytoskeleton and recently has been identified as a potent allergen in pollen. We examined the profilin gene family in the model plant, Arabidopsis thaliana, and found that it contained approximately 8 to 10 members. Four distinct profilin sequences, three cDNAs, PRF1, PRF2, and PRF3, and two genomic clones, PRF1 and PRF4, were isolated and characterized. These genes encoded four distinct profilin isoforms of 131 to 134 amino acids. Northern and reverse-transcriptase polymerase chain reaction analyses demonstrated that Arabidopsis PRF1 was expressed in all major plant organs, whereas PRF4 was specifically expressed in mature pollen. Gene trees constructed from amino acid sequence data revealed the presence of two ancient, distinct profilin gene classes in plants. PRF4 was in a class with previously identified pollen-specific profilins from monocot and dicot species. PRF1, PRF2, PRF3, and a distant dicot sequence formed a separate novel class, suggesting an ancient separation of plant profilins based on regulation and perhaps function. The coevolution of plant actin and profilin classes with similar patterns of expression is discussed. The similarity of plant, fungal, protist, insect, and nematode profilins and their extreme divergence from the vertebrate profilins has striking implications for the evolution of fungal-spore- and plant-pollen-profilins as allergens.

Allergens↗

PubMed1998

Localization of actobindin, profilin I, profilin II, and phosphatidylinositol-4,5-bisphosphate (PIP2) in Acanthamoeba castellanii.

Specific polyclonal antisera were raised against purified Acanthamoeba actobindin and synthetic peptides corresponding to regions of maximum charge differences in Acanthamoeba profilin I and profilin II. Immunofluorescence studies with these antibodies showed profilin I to be distributed throughout the Acanthamoeba cytoplasm, except for lamellipodia, with the highest fluorescence intensity in cortical regions in which monomeric actin also was present, as shown by labeling with fluorescent DNase. In contrast, profilin II appeared to be uniformly associated with the plasma membrane except at sites of pseudopod extension, where the concentration was frequently decreased, in addition to cortical regions. Immunofluorescence studies using a monoclonal antibody specific for phosphatidylinositol-4,5-bisphosphate (PIP2) suggested that its distribution is mostly limited to the plasma membrane. In contrast to the distribution of profilin II, PIP2 immunofluorescence was prominent at the leading edge of cells, including the plasma membrane of lamellipodia. Quantitative immunoelectron microscopy showed that profilin II was approximately 36 times more likely to localize to the plasma membrane than profilin I. Immunofluorescence and confocal microscopy localized actobindin to the base of lamellipodia. The differential localization of the three actin monomer-binding proteins suggests that they have different biologic functions in Acanthamoeba and is consistent with the hypotheses that (1) profilin I functions predominantly as an actin monomer-binding protein; (2) profilin II regulates, or is regulated by, PIP2; and (3) actobindin inhibits nucleation of new filaments and facilitates elongation of existing polarized filaments in actively motile regions.

Acanthamoeba↗

PubMed1995

Molecular cloning and characterization of profilin from tobacco (Nicotiana tabacum): increased profilin expression during pollen maturation.

Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA coding for tobacco profilin, which shared an average sequence identity of 75% with other plant profilins, was isolated from a tobacco pollen cDNA library by antibody screening. Tobacco profilin was expressed in Escherichia coli and purified by affinity to poly-(L-proline) Sepharose. A rabbit antiserum was raised against recombinant tobacco profilin and used to estimate the amount of profilin expressed in different tobacco tissues. Profilin can be detected in different somatic tissues, but the expression is 50-100 fold higher in mature pollen. Immunofluorescence and confocal laser scanning microscopy showed a homogeneous distribution of profilin in the cytoplasm of in vitro cultured pollen grains and pollen tubes of tobacco whereas some growing pollen tubes were stained more intensively a their tip. A possible role of pollen profilin as a developmentally upregulated microfilament precursor in mature pollen is discussed.

Actins↗

PubMed1991

Profilin and profilin mRNA in the cerebellum of the developing rat.

Profilin and profilin mRNA concentrations were determined in the cerebellum of the developing rat. Profilin represented nearly 0.1% of total Triton-soluble protein both at birth and at the end of the developmental period. During brain maturation, the profilin concentration was regulated at the transcriptional level. A transient increase of profilin mRNA and profilin was observed at the end of the first postnatal week, a period of intense cell proliferation, neuritic outgrowth and synaptogenesis. We propose that profilin may act by forming, during the critical period of cerebellar development, a reserve pool of monomeric actin that can easily be mobilized in cell regions such as growth cones or synaptic junctions where filamentous actin is highly concentrated.

Actins↗

PubMed1983

The profilin--actin complex: further characterization of profilin and studies on the stability of the complex.

Two forms of profilin can be isolated from calf spleen profilactin by chromatography on phosphocellulose. They can be distinguished by C-terminal analysis, which suggests that one of them lacks the C-terminal tyrosine and the penultimate glutamine residue. This is confirmed by treatment of profilin (+Tyr) with carboxypeptidase A, which removes the C-terminal tyrosine (rapidly) and the penultimate glutamine residue (slowly), and thereby converts it to the other form as judged by chromatography on phosphocellulose. The two forms of profilin differ also in solubility and in mobility during so-called 'charge shift' electrophoresis, indicating differences in their ability to bind detergents. Recombination studies using profilin with or without a modified C-terminus demonstrated that this part of profilin is relatively unimportant for the interaction with actin. On the other hand, experiments with native and modified actin revealed that the C-terminus of actin is of the utmost importance for the stability of the profilactin complex. Analysis of the u.v. absorbance and far-u.v. circular dichroism spectra of profilin and actin did not reveal any major changes in the conformation of the proteins accompanying the modifications at the C-terminal ends. Finally, it is reported that purified profilactin contains variable amounts of a protein factor which causes an apparent stabilization of profilactin in solution.

Actins↗

PubMed1990

Purification of profilin from Saccharomyces cerevisiae and analysis of profilin-deficient cells.

We have isolated profilin from yeast (Saccharomyces cerevisiae) and have microsequenced a portion of the protein to confirm its identity; the region microsequenced agrees with the predicted amino acid sequence from a profilin gene recently isolated from S. cerevisiae (Magdolen, V., U. Oechsner, G. Müller, and W. Bandlow. 1988. Mol. Cell. Biol. 8:5108-5115). Yeast profilin resembles profilins from other organisms in molecular mass and in the ability to bind to polyproline, retard the rate of actin polymerization, and inhibit hydrolysis of ATP by monomeric actin. Using strains that carry disruptions or deletions of the profilin gene, we have found that, under appropriate conditions, cells can survive without detectable profilin. Such cells grow slowly, are temperature sensitive, lose the normal ellipsoidal shape of yeast cells, often become multinucleate, and generally grow much larger than wild-type cells. In addition, these cells exhibit delocalized deposition of cell wall chitin and have dramatically altered actin distributions.

Actins↗

PubMed1993

Cloning and expression of a novel human profilin variant, profilin II.

We have isolated a 1.7 kbp cDNA encoding a 140 amino acid protein (15.1 kDa, pI 5.91) with a high sequence similarity (62%) to human profilin (profilin I). We have termed this variant profilin II. Northern blot analysis showed that profilin II is highly expressed in brain, skeletal muscle and kidney and less strongly in heart, placenta, lung and liver. In addition, three different transcript lengths were detected. Only one transcript of profilin I was found. The expression level of this was low in brain and skeletal muscle, medium in heart and high in placenta, lung, liver and kidney.

Amino Acid Sequence↗

PubMed1988

The primary structure of human platelet profilin: reinvestigation of the calf spleen profilin sequence.

The primary structure of human platelet profilin was determined by aligning the sequences of its tryptic peptides to the previously determined calf spleen profilin sequence [(1979) FEBS Lett. 101, 161-165]. Comparison of the peptide fingerprints of the two proteins suggested a higher homology than that found by direct sequence comparison. We therefore reinvestigated the sequences of the peptides from calf spleen profilin. We identified four incorrect charge assignments and a deletion of three residues. The similarity between the two vertebrate profilins amounts to 95%.

Amino Acid Sequence↗

PubMed1991

Dictyostelium discoideum contains two profilin isoforms that differ in structure and function.

Two profilin isoforms (profilins I and II) have been purified from Dictyostelium discoideum, using affinity chromatography on a poly(L-proline) matrix; the isoforms could be separated by cation-exchange chromatography on a FPLC system. The gene coding for profilin I was cloned from a lambda gt11 cDNA library using a profilin I-specific monoclonal antibody. The profilin II cDNA was isolated by probing the cDNA library with an oligonucleotide deduced from the N-terminal amino acid sequence of profilin II, which has an open N terminus in contrast to profilin I. The deduced amino acid sequences of both genes show that profilin I in comparison to profilin II is slightly larger (13,064 Da vs 12,729 Da), has a more acidic isoelectric point (calc. pI 6.62 vs 7.26) and shares with profilin II 68 identical residues out of 126 amino acids. Although both profilins contain a conserved lysine residue in the putative actin-binding region and can be crosslinked covalently to G-actin, the crosslinking efficiency of profilin II to actin is substantially higher than that of profilin I. These data are in agreement with studies on the functional properties of the profilin isoforms. In most preparations profilin II was more efficient in delaying the onset of elongation during the course of actin polymerization and caused a higher critical concentration for actin polymerization than profilin I, probably due to the slightly increased affinity of profilin II for D. discoideum G-actin (approx. Kd 1.8 x 10(-6) M) as compared to that of profilin I (approx. Kd 5.1 x 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1997

Differential colocalization of profilin with microfilaments in PtK2 cells.

Profilins are thought to be involved in the control of actin dynamics in eukaryotic cells. In accordance with this concept, profilin was found to be colocalized with the cortical microfilament webs in leading lamellae of locomoting and spreading fibroblasts. However, so far, there is little information on the distribution of profilin in other cell types. In this study, we report on the colocalization of profilin with various microfilament suprastructures in the epithelial cell line PtK2. This cell line, which is derived from rat kangaroo, contains a profilin sharing an N-terminal epitope with bovine and human profilin I, as seen by immunoblotting with monoclonal antibodies. By using immunofluorescence in conjunction with conventional fluorescence microscopy and confocal laser-scanning microscopy, we found profilin in ruffling areas of the peripheral lamellae and nascent stress fibers of spreading cells, whereas the peripheral belts of stationary cells growing in epithelioid sheets lacked profilin staining. In these cells, profilin was primarily distributed in a fine reticular or vesicular network that was not related to the microfilament system. Conspicuously low levels of profilins was not related to the contractile ring of mitotic cells. This was found for different fixation protocols and antibodies of the IgG and IgM type, respectively, indicating that lack of staining of the cleavage furrow was not due to antibody penetration problems. Depending on the fixation protocol, the nuclear matrix appeared strongly positive or negative for profilin. Cells microinjected with birch pollen profilin and labeled with a birch profilin-specific monoclonal antibody corroborated the results obtained with the endogeneous protein: The injected profilin was targeted to the cortical web and to nascent stress fibers of spreading cells but not to the cleavage ring of mitotic cells. These results suggest that high concentrations of a profilin I homologue are preferentially located with those microfilament suprastructures in PtK2 cells that are subject to rapid modulation by external signals.

Actin Cytoskeleton↗

PubMed1989

Characterization of renatured profilin purified by urea elution from poly-L-proline agarose columns.

We present evidence that native profilin can be purified from cellular extracts of Acanthamoeba, Dictyostelium, and human platelets by affinity chromatography on poly-L-proline agarose. After applying cell extracts and washing the column with 3 M urea, homogeneous profilin is eluted by increasing the urea concentration to 6-8 M. Acanthamoeba profilin-I and profilin-II can subsequently be separated by cation exchange chromatography. The yield of Acanthamoeba profilin is twice that obtained by conventional methods. Several lines of evidence show that the profilins fully renature after removal of the urea by dialysis: 1) dialyzed Acanthamoeba and human profilins rebind quantitatively to poly-L-proline and bind to actin in the same way as native, conventionally purified profilin without urea treatment; 2) dialyzed profilins form 3-D crystals under the same conditions as native profilins; 3) dialyzed Acanthamoeba profilin-I has an NMR spectrum identical with that of native profilin-I; and 4) dialyzed human and Acanthamoeba profilins inhibit actin polymerization. We report the discovery of profilin in Dictyostelium cell extracts using the same method. Based on these observations we conclude that urea elution from poly-L-proline agarose followed by renaturation will be generally useful for preparing profilins from a wide variety of cells. Perhaps also of general use is the finding that either myosin-II or alpha-actinin in crude cell extracts can be bound selectively to the poly-L-proline agarose column depending on the ionic conditions used to equilibrate the column. We have purified myosin-II from both Acanthamoeba and Dictyostelium cell extracts and alpha-actinin from Acanthamoeba cell extracts in the appropriate buffers. These proteins are retained as complexes with actin by the agarose and not by a specific interaction with poly-L-proline. They can be eluted by dissociating the complexes with ATP and separated from actin by gel filtration if necessary.

Acanthamoeba↗